iii. metabolism - glycolysispeople.uleth.ca/~steven.mosimann/bchm3300/bchm3300_l4.pdf · iii....

Biochemistry 3300 Slide 1

III. Metabolism

- Glycolysis

Department of Chemistry and BiochemistryUniversity of Lethbridge

Biochemistry 3300


Major Pathways of Glucose Utilization

These three pathways are the most significant in terms of the amount of glucosethat flows through them in most cells.


The Two Phases of Glycolysis

Breakdown of the glucose (6C) into two molecules of the pyruvate (3C) occurs in ten steps.

Ten steps of Glycolysis can besubdivided in two Phases:

I. The Preparatory Phase (steps 1-5)- spend ATP- glucose → 2 glyceraldehyde-3-phosphate

II. The Payoff Phase (steps 6-10)- generate ATP & NADH- 2 glyceraldehyde-3-phosphate → 2 pyruvate


Preparatory Phase of Glycolysis

Enzymes- 2 Kinases- 2 Isomerases

- 1 Aldolase


Payoff Phases of Glycolysis

Enzymes- 2 Kinases- 1 Mutase

- 1 Dehydrogenase- 1 Enolase


Yield (in energy equivalents) per glucose

Glucose + 2NAD+ + 2ADP + 2Pi

→ 2 pyruvate + 2NADH + 2H+ + 2ATP + 2H2O

Glucose + 2NAD+ → 2 pyruvate + 2NADH + 2H+ ∆G’1

O= -146 kJ/mol

Formation of 2x pyruvate, NADH and H+ (energy released):

Formation of 2 ATP (energy cost):

2ADP + 2Pi → 2ATP + 2H

2O ∆G’

2

O= 61.0 kJ/mol

∆G’S

O= ∆G’1

O + ∆G’2

O = -85 kJ/mol

Chemical equation for glycolysis:


A Historical Perspective

• 1854-1864: L. Pasteur establishes fermentation is caused by microorganisms.• 1897: E. Buchner demonstrates cell-free yeast extracts carry out this process.• 1905-1910: Arthur Harden and William Young discovered:

– Inorganic phosphate is required for fermentation and is incorporated into fructose-1-6-bisphosphate

– A cell-free yeast extract has a nondialyzable heat-labile fraction (zymase) and a dialyzable heat-stable fraction (cozymase).

• By 1940: Elucidation of complete glycolytic pathway (Gustav Embden, Otto Meyerhof, and Jacob Parnas).

Otto Fritz Meyerhof & Archibald Vivian Hill The Nobel Prize in Physiology or Medicine 1922


Glycolysis∆G

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Pathway coordinate


Hexokinase: First ATP Utilization

Reaction 1 : Transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate (G6P)

∆G’° = -16.7 kJ/molIrreversible under cellular conditionsdue to large, negative ∆G'


Hexokinase: First ATP Utilization

The two domains (grey/green) swing together when bound to Pi

- excludes H2O from active site (eg. electrostatic catalysis)

E + S ES complex


Phosphohexose Isomerase

∆G’° = 1.7 kJ/mol

Reaction 2: Phosphohexose isomerase catalyzes the conversion of G6P to F6P(ie. aldose to ketose isomerisation)

Isomerases and mutasestypically catalyze reactions with small ∆G'º (and ∆G')


Phosphohexose Isomerase

Overall reactioninvolves multiple (4)elementary steps

Acid-base catalysis


PFK-1: Second ATP Utilization

Reaction 3: Phosophofructokinase-1 (PFK-1) phosphorylatesfructose-6-phosphate (F6P)

PFK-1 plays a central role in control of glycolysis as it catalyzes one of the pathway’s rate-determining reactions.

∆G’° = -14.2 kJ/mol

Irreversible under cellular conditionsdue to large, negative ∆G'


Aldolase

Reaction 4: Aldolase catalyzes cleavage of fructose-1,6-bisphosphate (FBP)

∆G’° = 23.8 kJ/mol

How is the large, unfavourable ∆G’° for this reaction overcome?

The [Product]/[Substrate] is kept very small

Enzymes operating far from their equilibrium state are regulatory targets


Retro Aldol Reaction

How could (and does) aldolase enhance the rate of this reaction?

Stabilize the carbanion !

The aldolase mechanism is similar to the retro-aldol mechansim in organic chemistry


(Class I) Aldolase Reaction

Mechanism

Formation of a protonated Schiff's base (Lys 229) that stabilizes the carbanion/enamine

Class I Aldolase (Schiff's base mechanism)Class II Aldolase (Metalloenzyme)


Triose Phosphate IsomeraseReaction 5: Interconversion of the triose phosphates

Only G3P continues along the glycolytic pathway. DHAP(dihydroxyacetonephosphate) is rapidly and reversible converted to G3P by TPI (aka TIM)

∆G’° = 7.5 kJ/mol

Inhibitors used to study TPI mechanism


Triose Phosphate Isomerase

Ribbon diagram of TPI (TIM) in complex with its transition state analog2-phosphoglycolate.

Flexible loop (light blue) makes a hydrogen bond with the phosphate group of the substrate.

Removal of loop (mutagenesis) does not impair substratebinding but reduces catalytic rate by 105 fold.

stereoelectronic control(electrostatic catalysis)


Summary of Reaction 4 & 5

Fate of carbon atoms of glucose in the formation of glyceraldehyde-3-phosphate


'Cartoon' summary of preparatory phase of

glycolysis

Overall Reaction for Preparatory Phase of Glycolysis

Glucose + 2 ATP 2 Glyceraldehyde-3-phosphate + 2 ADP


The Payoff Phase


Glyceraldehyde-3-Phosphate Dehydrogenase

Reaction 6:Glyceraldehyde-3-phosphate dehydrogenase forms the first “high-energy” intermediate.

∆G’° = 6.3 kJ/mol

Substrate Level PhosphorylationP

i is the substrate !!

High energy phosphodiester and NADHproduced without expending ATP !!!


Glyceraldehyde 3-Phosphate DehydrogenaseReaction Mechanism

David TrenthamMechanism


Glyceraldehyde 3-Phosphate Dehydrogenase(covalent intermediate)

Covalent intermediate:

thiohemiacetals are higherenergy compounds than hemiacetals


Glyceraldehyde 3-Phosphate Dehydrogenase (oxidation step)

Bound NAD+ accepts electronsThat are funnelled intooxidative phosphorylation


Glyceraldehyde 3-Phosphate Dehydrogenase(substrate level phosphorylation)

Thioester is cleaved by Pi producing a high energy compound: 1,3-bisphosphoglycerate


Glyceraldehyde 3-Phosphate Dehydrogenase(product release)


Glyceraldehyde-3-Phosphate Dehydrogenase

Mechanistic Studies:

Iodoacetate inactivates enzyme by modification of active site cysteine

Aldehyde group provides H+ for NAD+ reduction

Pi is substrate


Phosphoglycerate Kinase: First ATP Generation

Glycolysis:

Two kinase enzymes (hexokinase and PGK)are homologs with different substrate specificities

Upon substrate binding, the two domains of PGK swing together, providing a water-free environment (just like hexokinase)


Phosphoglycerate Kinase: First ATP Generation

∆G’° = -18.5 kJ/mol

1,3-bisphosphoglycerate is a higherenergy compound than ATP so the reaction has a large favourable freeenergy change


Mechanism of PGK reaction

Phosphotransferase reactionsimilar to hexokinase (and PFK)


Energetics of the Glyceraldehyde-3-Phosphate Dehydrogenase:Phosphoglycerate Kinase

Reaction Pair

Coupling the two steps of the pathway: Under standard condition: 1,3-BPG phosphotransfer drives the coupled reaction forming NADH and ATP.


Phosphoglycerate Mutase

Reaction 8: Catalyzes a reversible shift of the phosphoryl groupbetween C-2 and C-3 of glycerate; Mg2+ and 2,3-bisphosphoglycerate are essential.

∆G’° = 4.4 kJ/mol


Reaction Mechanism of PGM

Catalytic amounts of 2,3-bisphosphoglycerate are required for enzymatic activity.

2,3-bisphosphoglycerate 'activates' PGM by phosphorylating active site histidine

ie. Enzyme is inactive until phosphorylated


Reaction Mechanism of PGM

Step 1:

Phosphohistidine transfersphosphoryl to 3PG forming2,3-BPG

Step 2:

2,3-BPG transfers phosphoryl(3C) to His forming 2BPG andphosphorylated His


Gycolysis Influences Oxygen Transport

2,3-BPG binds to deoxyhemoglobin and alters (decreases)oxygen binding affinity.

Erythrocytes synthesize and degrade 2,3-BPG using a 'detour' within the glycolytic pathway.

Requires two enzymes (only) expressed in Erythrocytes


Gycolysis Influences Oxygen Transport

Lower [2,3-BPG] in erythrocytes resulting from hexokinase-deficiencyresults in increased hemoglobin oxygen affinity.

Higher [2,3-BPG] in erythrocytes resulting from PK-deficiencyresults in decreased hemoglobin oxygen affinity.


Enolase: Second “High Energy” Intermediate

∆G’° = 7.5 kJ/mol


Enolase Reaction Mechanism

Can be inhibited by F-. Why?F- binds to strongly metals

Overall reaction is a dehydration


Enolase Reaction Mechanism

Structure of enolasecatalytic center(PDB ID 1ONE)


Pyruvate Kinase : Second ATP Generation

Not a homolog of other kinases inglycolysis

∆G’° = -31.4 kJ/mol

Irreversible under cellular conditionsdue to large, negative ∆G'


Pyruvate Kinase :Second ATP Generation

Tautomerization ofenolpyruvate to pyruvate.

Phosphotransfer to ADP


The Payoff Phase

4 ATP2 NADH

Overall: glycolysisgenerates 2 ATP and2 NADH


Summary of Enzyme Properties

Reactions with large G' tend to be regulatory targets (Red)

Enzyme EC Class Mechanism Intermediate G'º G' Features

Hexokinase Transferase Phosphotransferase ATP → ADP

- -17 -27 Costs ATP

G6P Isomerase Isomerase Aldose → Ketose enol 2 -1

Phosphofructokinase Transferase Phosphotransferase ATP → ADP

- -14 -26 Costs ADP

Aldolase Lyase Schiffs base eneamine 23 -6 6C sugar → 2x 3C sugar

Triose Phosphate Isomerase Isomerase Ketose → Aldose enol 8 0

G3P Dehydrogenase Oxidoreductase NAD+ → NADH; SLP thioester 6 0 1,3-BPG ( Substrate level phosphorylation); NADH

Phosphoglycerate Kinase Transferase Phosphotransferase ADP → ATP

- -18 -1 Generates ATP

Phosphoglycerate Mutase Isomerase Phosphate migration 2,3-BPG 4 -1

Enolase Lyase Dehydration; Mg2+ enediol 8 -2 PEP

Pyruvate Kinase Transferase Phosphotransferase ADP → ATP

- -31 -14 a-keto -CO2- ; generates ATP

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